Respiratory virus infection caused by enveloped RNA viruses, such as parainfluenza and respiratory syncytial viruses, is a major public health concern. These viruses infect and replicate in the upper and/or lower respiratory tracts and transmit through airways. These respiratory viruses bud from the apical surface of polarized epithelial cells. Obviously, efficient virus assembly in respiratory epithelial cells is a key factor for virus transmission and pathogenicity. However, the molecular mechanism of virus assembly, especially how viral structural components are transported to the plasma membrane assembly/budding sites is poorly understood, partially due to lack of a method to monitor trafficking of viral components in live infected cells. In this proposal, we will use Sendai virus (SeV), one of the best-characterized paramyxoviruses, to reveal how viral nucleocapsids (vRNPs) are transported to assembly sites. Using the reverse genetics system, we successfully rescued a recombinant SeV (rSeV-LeGFP) whose L protein is tagged with enhanced green fluorescent protein (eGFP). This virus allows us to monitor real time trafficking of the vRNP in live infected cells. Using the virus, we successfully recorded vRNP movement through microtubules in infected cells. Additional data strongly suggest that the intracellular vesicular trafficking pathway regulated by Rab11a is involved in vRNP translocation. Taking full advantage of rSeV-LeGFP and our ability to rescue various SeV mutants, we propose to unveil the cellular and viral factors involved in the process of vRNP trafficking and virus assembly in respiratory epithelial cells. We will identify the cellular machinery and proteins responsible for vRNP transport, as well as the viral proteins that mediate the process. In this project, we will uncover the process of assembly of SeV and related human respiratory paramyxoviruses, especially the role of the intracellular vesicular trafficking pathway in vRNP transport and assembly of virions at the apical surface budding sites of epithelial cells. This research will shed light on the unknown critical process of respiratory virus assembly, which is expected to provide valuable data to develop safe attenuated vaccines or antiviral agents.